An enormous range of medicinally important polyketide and peptide natural products assembled by modular polyketide synthases (PKSs), non-ribosomal peptide synthetases (NRPS) and mixed PKS/NRPS systems have macrocyclic structures, including the antibiotics erythromycin (PKS) and daptomycin (NRPS), the immunosuppressants cyclosporin (NRPS) and rapamycin (PKS/NRPS) and the antitumor agent epothilone (PKS/NRPS). PKSs and NRPSs are very large multifunctional proteins that are organized into sets of functional domains termed modules (Cane et al, Science (1998) 282:62-8; Marahiel et al, Chem. Rev. (1997) 97:2651-74). The sequence of modules corresponds directly to the structure of the product. Partially formed products are covalently tethered by thioester linkages to a carrier protein domain in each module. The thiol tether on each carrier domain is phosphopanetheine, which is attached to a conserved serine residue in the carrier protein in a post-translational priming reaction catalyzed by phosphopanetheinyl transferase (Lambalot et al, Chem. Biol. (1996) 3:923-36). Chain initiation involves loading a specific monomer onto each carrier protein's thiol tether. Subsequent chain elongation steps involve transfer of the growing chain from an upstream carrier protein to the adjacent downstream carrier protein-bound monomer. The full-length chain is almost always cyclized and released from the enzyme at the C-terminus of the NRPS or PKS system by a 28-35 kD TE domain (Cane et al, Science (1998) 282:62-8). During this final cyclization step, deacylation of the resulting acyl-O-TE intermediate at the C-terminal TE domain occurs either by intramolecular cyclization to form macrolactones or macrolactams or by hydrolysis.
The 6-deoxyerythronolide B synthase (DEBS) protein is a multidomain PKS protein with an integral TE domain that catalyzes cyclization of a protein-bound polyketide. Modification of domain identity or sequence in the natural DEBS protein by single or multiple domain substitutions or insertions of natural heterologous subunits generates DEBS protein variants that produce compounds with various ketide unit sequences. Systematic variation of the sequence of domains in the multidomain DEBS can in principle generate libraries of compounds (McDaniel et al, PNAS, (1999) 96:1846-51; McDaniel et al, Chem Biol, (2000) 7:77-84).
Kao disclosed the design and construction of engineered derivatives of the DEBS protein that is capable of synthesizing 6 and 8 member-ring lactones. The engineered DEBS derivatives included systems with protein modules, e.g. domains, exclusively from the DEBS system and hybrid derivatives that included protein modules from both the DEBS system and from the rapamycin PKS (RAPS) protein system. The DEBS-only derivative generated 6-member lactones and the DEBS-RAPS hybrid catalyzed the formation of a new 8-member lactone (Kao, J. Am. Chem. Soc. (1997) 119:11339-40).
The expression of a naturally occurring amino-terminal truncated form of a PKS protein to generate a macrocyclic molecule with smaller rings is described by Xue (Xue et al, Nature, (2000) 403:571-5). Truncation of the last condensation module from PikAIV in S. venezuelae leads to ‘skipping’ of the final condensation cycle in polyketide biosynthesis to generate a 12-membered ring macrolactone, 10-deoxymethynolide, instead of the 14-membered ring product molecule, narbonolide.
Jacobsen et al disclosed a method for producing a series of polyketides by blocking the first condensation step of the DEBS protein system and introducing exogenous synthetic engineered molecules. The synthetic methods using the blocked DEBS protein system resulted in the highly selective production of a variety of polyketide molecules including aromatic and ring-expanded variants of 6-deoxyerythronolide B (Jacobsen et al, Science, (1997) 277:367-9).
The DNA sequence encoding the TE domain from 6-deoxyerythonolide B synthase (DEBS) has been excised and independently expressed and the domain isolated either as isolated TE domain enzyme (Gokhale, Chem Biol, (1999) 6:117-25) or as part of an ACP-TE di-domain protein (Aggarwal, J Chem Soc, Chem Comm, (1995) 15:1519-20). Thioester substrates were exclusively hydrolyzed to corresponding carboxylic acids by both the isolated TE domain and the ACP-TE didomain. The ACP-TE di-domain further hydrolyzes aryl esters. No cyclization was observed in these systems.
Many useful pharmaceuticals have macrocyclic structures (a large ring composed of 10 or more atoms). Traditional synthetic chemistry approaches to the synthesis of macrocyclic compounds have drawbacks including, but not limited to, low yields of macrocyclic molecule products, protecting groups required to block or mask reactive functionalities, and the need to carry out reactions in organic solvents.
International Publication No. WO 00/36093 describes a method for producing cyclic peptides and splicing intermediates of peptides in a looped conformation. The methods utilize the trans-splicing ability of split inteins to catalyze cyclization of peptides interposed between two portions of a split intein. The interaction of the two portions of the split intein creates a catalytically active intein, which catalyzes the formation and liberation of a cyclic peptide product.
However, there remains an unfulfilled need for synthetic methods for preparing macrocyclic molecules in high yield without requiring functional group protection or carrying out reactions in organic solvents.